(Hypertension. 1996;28:354-360.)
© 1996 American Heart Association, Inc.
Articles |
Use-Dependent Loss of Acetylcholine- and Bradykinin-Mediated Vasodilation After Nitric Oxide Synthase Inhibition
Evidence for Preformed Stores of Nitric Oxide–Containing Factors in Vascular Endothelial Cells
the Cardiovascular Center and Departments of Pharmacology (R.L.D., A.K.J., S.J.L.), Anesthesia (J.N.B.), and Psychology (A.K.J.), The University of Iowa, Iowa City.
Correspondence to Stephen J. Lewis, PhD, Department of Pharmacology, 2-210 Bowen Science Bldg, The University of Iowa, Iowa City, IA 52242.
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Abstract |
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In the present study, we examined the possibility that the endothelium-dependent vasodilators acetylcholine and bradykinin release preformed pools of nitric oxide–containing factors. Successive injections of selected doses of acetylcholine (1.18±0.3 µg/kg IV) or bradykinin (5 µg/kg IV) caused reproducible hypotensive and vasodilator responses within sympathetically intact and sympathetically denervated hindlimbs of conscious rats. After administration of the nitric oxide synthesis inhibitor N
-nitro-L-arginine methyl ester (L-NAME, 25 µmol/kg IV), the first injection of acetylcholine or bradykinin produced pronounced depressor and vasodilator responses that, in the case of bradykinin, were greater than those observed before L-NAME administration. However, each successive injection of acetylcholine and bradykinin produced progressively smaller responses, such that the later injections elicited a markedly diminished hypotension and vasodilation. This "use-dependent" loss of endothelium-dependent vasodilation was not due to the diminished vasorelaxant potency of nitric oxide–containing factors because the vasodilator effects of the nitric oxide donor sodium nitroprusside (32 µg/kg IV) and the S-nitrosothiol compound S-nitrosocysteine (200 nmol/kg IV) were augmented in the presence of L-NAME. These results suggest that the use-dependent loss of the hemodynamic effects of acetylcholine and bradykinin in L-NAME–treated rats may be due to the release and subsequent depletion of a factor whose synthesis depends on the bioavailability of nitric oxide. Taken together, these results suggest that preformed pools of nitric oxide–containing factors exist within the endothelium of resistance vessels and that endothelium-dependent agonists exert their vasorelaxant effects at least in part by the mobilization of these preformed pools.
Key Words: acetylcholine • bradykinin • endothelium-derived factor • hemodynamics • nitric oxide • rats
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Introduction |
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Furchgott and Zawadski1 reported that the endothelium plays an obligatory role in acetylcholine-induced relaxation of isolated blood vessels. Subsequently, Furchgott et al2 and Ignarro et al3 independently provided evidence that the endothelium-derived factor responsible for the acetylcholine-induced vasorelaxation was NO. Subsequent studies have demonstrated that vascular endothelial cells contain an NADPH-dependent flavin–containing enzyme (NO synthase) that catalyzes the conversion of L-arginine to NO4 and that NO synthesis inhibitors consistently cause a marked reduction of acetylcholine- and bradykinin-induced vasorelaxation in vitro.5 6 However, NO synthase inhibitors do not or only partially attenuate endothelium-dependent vasodilation in vivo.6 7 8 9 10 These findings suggest that the vasorelaxant effects of acetylcholine and bradykinin on the large vessels used in the in vitro studies involve the release of de novo synthesized NO or newly formed NOFs such as SNC11 or dinitrosyl iron (II) complexes,12 whereas the vasodilation in vivo may involve the release of additional factors from the endothelium of resistance vessels.
Ignarro13 has postulated that acetylcholine may trigger the exocytotic release of preformed vesicular pools of NOFs such as SNC from the vascular endothelium. At present, there is no definitive evidence that NOFs such as S-nitrosothiols or dinitrosyl iron (II) complexes exist in preformed pools within the plasmalemmal vesicles of endothelial cells.14 15 16 17 18 19 20 If preformed vesicular pools of NOFs exist in the vascular endothelium, then the vasodilator effects of endothelium-dependent agonists should progressively diminish upon their repeated administration in animals treated with an NO synthesis inhibitor. The "use-dependent" diminution of endothelium-dependent vasodilation would be due to the gradual depletion of these preformed pools of NOFs that could not be regenerated in the absence of NO synthesis. We have recently demonstrated that repeated episodes of medium-intensity electrical stimulation of the lumbar sympathetic chain produced pronounced and equivalent reductions in HLR in pentobarbital-anesthetized rats.21 After administration of the NO synthesis inhibitor L-NAME, the first episode of electrical stimulation produced a pronounced vasodilation. However, subsequent episodes of electrical stimulation produced progressively and markedly smaller vasodilator responses. These findings suggest that sympathetic neurogenic vasodilation might be mediated by the release of preformed NOFs. The vasodilation produced by the electrical stimulation of the lumbar chain might be due to the release of preformed NOFs from postganglionic NO synthase–positive lumbar sympathetic nerves22 or might involve the sympathetic nerve–derived, catecholamine-mediated release of preformed NOFs from the vascular endothelium.21
To more clearly demonstrate that preformed NOFs might exist in the vascular endothelium of resistance vessels, in this study we examined the effects of successive intravenous injections of selected doses of the endothelium-dependent vasodilators acetylcholine and bradykinin on MAP, MR (in the bradykinin study), and HLR in the intact (HLRi) and sympathetically denervated (HLRd) hindlimbs of conscious rats before and after injection of the NO synthesis inhibitor L-NAME (25 µmol/kg IV). We compared the effects of acetylcholine and bradykinin in intact and sympathetically denervated hindlimbs to remove the possible confounding influence of these agents on sympathetic transmission23 as well as baroreceptor-mediated reflex changes in sympathetic nerve activity that would occur in response to the acetylcholine- and bradykinin-induced falls in MAP. To determine whether changes in the vasodilator effects of acetylcholine or bradykinin in the L-NAME–treated rats were due to alterations in the potency of endogenous NOFs, we also examined the effects of the NO synthase inhibitor on the vasorelaxant effects of the NO donor SNP and SNC. These endothelium-independent vasodilators were given after the injections of acetylcholine or bradykinin in both the saline- and L-NAME–treated rats.
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Methods |
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Surgery
The protocols described below were approved by the University of Iowa Animal Care and Use Committee. One week before the experiments, male Sprague-Dawley rats (300 to 400 g) were anesthetized with ketamine (120 mg/kg IP) and acepromazine maleate (12 mg/kg IP) and surgically implanted with catheters in the left common carotid artery and jugular vein for measurement of pulsatile pressure, MAP, and heart rate and administration of drugs, respectively. Immediately after catheterization, a midline laparotomy was performed and rats underwent selective hindlimb vasculature denervation in which the left lumbar sympathetic chain was isolated, cut, and removed caudally to the bifurcation of the left common iliac artery and vein. The right sympathetic chain was left intact. In a sham-operated group of rats (n=5), the left sympathetic trunk was exposed but not transected. Miniature pulsed-Doppler flow probes were placed on the left and right iliac arteries for measurement of HLFi, HLFd, HLRi, and HLRd. In the bradykinin experiments, a Doppler flow probe was also placed on the superior mesenteric artery for measurement of mesenteric blood flow and MR. The probes were sutured in place, the leads and catheters were tunneled subcutaneously and exteriorized between the scapulae, and the wounds were closed. To protect the probe wires and polyethylene tubing while allowing rats unrestricted movement during recovery and experimental testing, we led the free ends of the catheters and Doppler leads through a stainless steel skin button–spring swivel assembly that was mounted to a ring stand clamp and suspended above the cage. The skin button was attached to the skin incision in the scapular region with stainless steel sutures. Rats were allowed 7 days of recovery before experimental testing. Details of the Doppler technique, including construction of the probes, the reliability of the method for the estimation of flow velocity, and quantitative determination of percent changes in vascular resistance, have been described previously.21 22 24
Protocols
In one group of rats (n=6), we determined the intravenous dose of acetylcholine that produced approximately 50% reduction in HLRi in each of the rats. These doses were 0.1, 1, 1, 1, 2, and 2 µg/kg (1.18±0.3 µg/kg IV). On the first day, these doses were given six times at 5-minute intervals before and after saline injection. In three of the rats, the first acetylcholine injection was given 15 minutes after saline administration (to coincide with the time at which the maximal L-NAME–induced changes in resting parameters had been established, see below), and then each subsequent acetylcholine injection was given at 5-minute intervals. In the other three rats, the first acetylcholine injection was given 45 minutes after saline administration (to coincide with the time of the last acetylcholine injection in the above experiment), and then each subsequent acetylcholine injection was again given at 5-minute intervals. Since the results of these experiments yielded virtually identical results, the data were pooled for statistical analysis. On the following day, these acetylcholine doses were given six times before and after L-NAME injection (25 µmol/kg IV). In three of the rats, the acetylcholine injection was given 15 minutes after L-NAME administration (the time at which the maximal L-NAME–induced changes in resting parameters had been established), and then each subsequent acetylcholine injection was given at 5-minute intervals. In the other three rats, the first acetylcholine injection was given 45 minutes after L-NAME administration (to coincide with the time of the last acetylcholine injection in the above experiment), and then each subsequent acetylcholine injection was again given at 5-minute intervals. These data were also pooled for statistical analysis. In addition, the acetylcholine dose was increased to 1, 2, 2, 2, 4, and 4 µg/kg in each rat, respectively (2.50±0.50 µg/kg IV; change, 1.32±0.22 µg/kg), and this dose was given three times at 5-minute intervals. The first of these injections was given 5 to 10 minutes after the final injection of the lower dose of acetylcholine.
In another group of rats (n=5), we examined the hemodynamic effects produced by three successive bradykinin injections (5 µg/kg IV), each given 5 minutes apart, before and after saline injection on the first day of the experimental protocol. On the second day, we examined the hemodynamic effects produced by three successive bradykinin injections (5 µg/kg IV), each given 5 minutes apart, before and after L-NAME injection (25 µmol/kg IV, n=5). In these experiments, the first bradykinin injections were given 15 (n=2) or 30 (n=3) minutes after saline or L-NAME injection. Again, since the results of these experiments yielded similar results, the data were pooled for statistical analysis. This bradykinin dose was selected for repeated administration because it caused reproducible hemodynamic effects and it is well below doses that activate nociceptive visceral afferents.25
To determine whether changes in the vasodilator effects of acetylcholine or bradykinin in the above rats were due to alterations in the potency of endogenous NOFs, we examined the effects of the NO donor SNP (32 µg/kg IV) and the S-nitrosothiol compound SNC (200 nmol/kg IV) before and after L-NAME or saline injection. SNP and SNC were given after acetylcholine or bradykinin before and after saline or L-NAME injection. To test that the sympathectomy was complete, we allowed rats 1 day to recover from the above experimental procedures and examined the effects of the 
1-adrenoceptor antagonist prazosin (100 µg/kg IV) on HLRi and HLRd.
Drugs
Sodium nitrite, acepromazine maleate, L-cysteine, L-NAME, acetylcholine, and bradykinin were obtained from Sigma Chemical Co. SNP was from Abbott Laboratories. Ketamine was from Aveco Co. Stock solutions of SNC were prepared just before use by reacting 1-mL solutions of 0.2 mol/L sodium nitrite (containing 100 µL of 1N HCl) and 0.2 mol/L solutions of L-cysteine. This resulted in a stable (pH approximately 3) 0.1 mol/L stock solution of L-SNC. The stock and test solutions of L-SNC were routinely examined spectrophotometrically11 to ensure that the concentrations of L-SNC were appropriate.
Statistics
Data are presented as mean±SE. The single SEM values displayed on each dose-response curve in the figures were determined by the formula SEM=(EMS/n), where EMS is the error mean square term from the ANOVA, and n is the number of rats per group. The data were analyzed by repeated measures ANOVA26 followed by Student's modified t test with the Bonferroni correction for multiple comparisons between means27 using the EMS term from the ANOVA.
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Results |
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Effects of Surgical Sympathectomy on Resting HLR
Before transection of the left lumbar sympathetic trunk, the resting left and right HLF values in the anesthetized rats were equivalent (3.4±0.7 versus 3.8±0.6 kHz, respectively; n=16, P>.05). Seven days later, the resting left and right HLR values of the conscious sham-operated rats were equivalent (34±4 versus 38±5 mm Hg/kHz, n=5, P>.05). Table 1
summarizes the effects of chronic surgical transection of the lumbar sympathetic chain on HLR and the effects of the selective 1-adrenoceptor antagonist prazosin (100 µg/kg IV, n=5) or the NO synthesis inhibitor L-NAME (25 µmol/kg IV, n=11) on resting hemodynamic parameters in these conscious rats. Seven days after surgery, resting HLRd was markedly higher than HLRi (pre-HLRd versus pre-HLRi values, P<.05). Prazosin produced sustained falls in MAP and HLRi but did not effect HLRd. L-NAME produced sustained increases in MAP and HLRi but also did not effect HLRd.
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Effects of Repeated Injections of Acetylcholine and Bradykinin on Hemodynamic Parameters
A typical example of the effects of three successive injections of acetylcholine (0.1 µg/kg IV) on hemodynamic parameters of a conscious rat before and after L-NAME administration (25 µmol/kg IV) is shown in Fig 1. Before L-NAME administration, each acetylcholine injection produced identical decreases in MAP and increases in HLFi and HLFd. The acetylcholine-induced increases in HLFd were markedly smaller than those in HLFi. After L-NAME administration, the first acetylcholine injection produced pronounced decreases in MAP and increases in HLFi and HLFd. However, each successive injection produced markedly smaller responses. Fig 2 summarizes these data for acetylcholine. Before L-NAME administration, acetylcholine (1.18±0.3 µg/kg IV, n=6) produced pronounced reductions in MAP and HLRi but substantially smaller decreases in HLRd. Each successive acetylcholine injection produced similar decreases in these parameters. After L-NAME administration, the first dose of acetylcholine produced similar falls in MAP and HLRi compared with before the injection of the NO synthesis inhibitor, whereas the fall in HLRd was markedly exaggerated. However, each successive acetylcholine injection produced progressively smaller reductions in all three parameters.
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P<.05, after vs before L-NAME; *P<.05, subsequent injections vs first injection of acetylcholine; #P<.05, final vs second injection of acetylcholine.The initial administration of a higher dose of acetylcholine (2.50±0.50 µg/kg IV), given 5 to 10 minutes later, produced marked reductions in MAP, HLRi, and HLRd. However, subsequent injections produced progressively and markedly smaller responses. The effects of the three injections of acetylcholine were, for MAP, -30±6%, -19±4%, and -12±2%, respectively (P<.05, third injection versus first injection); for HLRi, -46±4%, -24±2%, and -14±3%, respectively (P<.05, second and third injections versus first injection); and for HLRd, -28±3%, -6±4%, and -2±2%, respectively (P<.05, second and third injections versus first injection). On the previous day, we established that six injections of acetylcholine (1.18±0.3 µg/kg IV) in these rats produced similar hemodynamic responses before and after saline injection, whereas in another group of saline-treated rats (n=6), we established that the repeated administration of higher doses of acetylcholine (3 µg/kg IV) also produce highly reproducible responses (data not shown).
Fig 3 summarizes the hemodynamic responses produced by three injections of bradykinin (5 µg/kg IV) before and after L-NAME administration (25 µmol/kg IV) in a separate group of conscious rats (n=5). Before L-NAME administration, this bradykinin dose produced an initial fall in MAP and MR but no changes in HLRi or HLRd. Small but significant falls in HLRi and HLRd subsequently occurred. Each bradykinin injection produced similar initial and subsequent hemodynamic responses. In addition, each bradykinin injection produced a similar arousal response. After L-NAME administration, the first bradykinin injection produced falls in MAP and MR that were equivalent to those observed before the NO synthesis inhibitor. However, bradykinin now produced marked reductions in HLRi and HLRd. Each successive bradykinin injection produced progressively smaller falls in MAP, HLRi, and HLRd. Although each of the three bradykinin injections produced similar peak falls in MR, the duration of each of these responses was significantly (P<.05) reduced after L-NAME (38±7%, 44±9%, and 48±11% of pre–L-NAME values). In addition, fourth, fifth, and sixth injections of bradykinin produced progressively smaller vasodilator responses in the mesenteric bed. The percent changes in MR produced by the first to the sixth injections of bradykinin were -53±8%, -49±6%, -45±6%, -34±4%, -27±3%, and -21±4%, respectively (P<.05, comparing the fifth and sixth injections with the first injection). Despite the use-dependent loss of the vasodilator effects of bradykinin in these L-NAME–treated rats, we noted that each bradykinin injection elicited arousal responses that were similar to those observed before L-NAME administration. On the previous day, it was established that three bradykinin injections (5 µg/kg IV) produced similar hemodynamic and behavioral responses before and after saline injection (data not shown).
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Hemodynamic Effects of SNP and SNC
To investigate whether the progressive loss of endothelium-dependent vasodilation was due to the diminished activity of NOFs, we determined the hemodynamic responses produced by administration of the NO donor SNP (32 µg/kg IV) and the S-nitrosothiol compound SNC (200 nmol/kg IV) before and after L-NAME administration in the rats that received acetylcholine and bradykinin. SNP and SNC were injected immediately after acetylcholine or bradykinin, before and after L-NAME. Table 2 summarizes the hemodynamic effects of SNP and SNC before and after injection of either saline or L-NAME in the 11 rats used in this study. SNP produced falls in MAP and HLRd but no changes in HLRi. Higher doses of SNP (64 to 128 µg/kg IV) did not produce greater reductions in MAP or HLRd or induce a relaxation in the innervated hindlimb bed (data not shown). In contrast, SNC produced pronounced falls in HLRi as well as in MAP and HLRd. The hemodynamic effects of SNP and SNC were similar before and after saline injection. In the presence of L-NAME, SNP produced exaggerated reductions in MAP and HLRd and was a potent vasodilator in the innervated bed. The SNC-induced falls in MAP and HLRi were also augmented after L-NAME administration. The augmented responses to SNP and SNC were due to L-NAME rather than to the use-dependent loss of endothelium-dependent vasodilation because a virtually identical augmentation of the hemodynamic effects of SNP and SNC was observed in rats that received L-NAME only (data not shown).
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Discussion |
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The principal finding of this study is that successive injections of selected doses of the endothelium-dependent agonists acetylcholine and bradykinin produce progressively smaller hypotensive and vasodilator responses in conscious rats pretreated with the NO synthesis inhibitor L-NAME. This use-dependent loss of acetylcholine- and bradykinin-mediated vasodilation was not due to a diminution in the potency of NOFs because the vasorelaxant effects of the NO donor SNP and the S-nitrosothiol compound SNC were exaggerated in the presence of L-NAME. These findings suggest that acetylcholine and bradykinin may cause the release of preformed pools of endothelium-derived factors whose synthesis requires the incorporation of NO into their structures. Moreover, the progressive diminution of endothelium-dependent vasodilation in the L-NAME–treated rats would be consistent with the gradual use-dependent depletion of these preformed pools of NOFs that cannot be regenerated in the absence of NO synthesis. In preliminary experiments, we found that L-arginine restored the hypotensive effects of acetylcholine in L-NAME–treated rats. We examined the effects of L-arginine (a bolus of 250 µmol/kg IV followed by infusion of 25 µmol/kg per minute IV) in three L-NAME (25 µmol/kg IV)–treated conscious rats. The sixth injection of acetylcholine (1 µg/kg IV) produced a fall in MAP of -7±3% before L-arginine administration. Within 15 to 20 minutes of L-arginine administration, the response to acetylcholine was -34±5% (P<.05 compared with the response before L-arginine administration). This later response was not different from that produced by acetylcholine before L-NAME administration (-37±6%, P>.05).
The increased biological effectiveness of NOFs may explain why the vasodilator effects of the first injection of acetylcholine and bradykinin were exaggerated in the presence of L-NAME. More specifically, acetylcholine and bradykinin may release preformed pools of NOFs that are able to produce an exaggerated vasodilation, which may be partly due to the upregulation of soluble guanylate cyclase in the vascular smooth muscle.28 Since the formation of these endothelium-derived factors apparently requires NO synthesis, these factors may be an S-nitrosothiol compound such as SNC11 or a dinitrosyl iron (II)–thiol complex.12 The possibility that the endothelium-derived relaxing factor responsible for the vasodilator actions of acetylcholine and bradykinin in the hindlimb vasculature may be an S-nitrosothiol rather than NO per se is supported by our observation that whereas SNC was a potent vasodilator in the hindlimb of conscious rats, the NO donor SNP produced only minimal changes in resistance in this bed. In addition, the exaggerated effects of the first dose of bradykinin in the L-NAME–treated rats might involve the enhanced release or biological actions of cyclooxygenase products such as prostacyclin. More specifically, a reduction in basal NO production by L-NAME might alter the activity of cyclooxygenase in response to bradykinin and thus contribute to the exaggerated vasodilation produced by the first injection of the autacoid.
There is now substantial evidence that S-nitrosothiols, dinitrosyl iron (II)–thiol complexes, and related NOFs can be formed and released from vascular endothelial cells,11 bronchial tissue,29 and activated macrophages.30 Although it is generally accepted that these NOFs are immediately released on their formation, Ignarro13 postulated that the endothelium might contain preformed vesicular pools of S-nitrosothiols and that endothelium-dependent agonists might release these stores by Ca2+-dependent exocytosis. This hypothesis is attractive because NO synthesis inhibitors do not or only partially inhibit endothelium-dependent vasodilation in vivo.6 7 8 9 10 The existence of preformed pools of NOFs within endothelial cells may explain why NO synthase inhibitors do not block the initial endothelium-dependent fall in vascular resistance in vivo, whereas they do diminish the duration of this vasodilation.6 7 8 9 10 Consequently, endothelium-dependent agonists may initiate vasodilation by causing the release of preformed pools of NOFs, whereas the vasorelaxation may be sustained by the subsequent release of newly synthesized NO or NOFs. The tonic Ca2+-dependent release of vesicular pools of NOFs from the vascular endothelium might be regulated by sympathetic nerve input,31 shear stress,13 and circulating hormones.32 The tonic release of these preformed pools of NOFs in the absence of NO synthesis would lead to the gradual depletion of these pools, and this would contribute to the increases in peripheral vascular resistances produced by NO synthesis inhibitors.
It is not widely recognized that a large population of plasmalemmal vesicles is the most conspicuous ultrastructural feature of endothelial cells.14 15 16 17 18 Electron microscopic studies have demonstrated that these vesicles form branching invaginations in the plasma membranes of endothelial cells.19 Moreover, there is evidence that these vesicles store water-soluble macromolecules.20 These findings suggest that the Ca2+-dependent exocytotic release of biologically active factors may occur in endothelial cells. Although it has not been established that NOFs exist within these plasmalemmal vesicles, it has been reported that endothelium-dependent relaxations are reduced by agents that inhibit mitochondrial transport or uncouple oxidative phosphorylation.33 These findings suggest that the production or release of endothelium-derived NOFs depends on mitochondrial synthesis of ATP. This is an important observation because there is no evidence that NO synthase is an ATP-dependent enzyme. Moreover, it is well established that Ca2+-dependent exocytotic release of vesicular stores of neurotransmitters and neurohormones from nerve terminals and the adrenal glands is strictly an ATP-dependent process.34 These findings lend support to the possibility that endothelium-dependent agonists may cause the exocytotic release of vesicular pools of NOFs. As mentioned, there is at present no definitive evidence that preformed pools of NOFs exist in the vascular endothelium. However, there is considerable evidence that vascular smooth muscle contains such preformed pools.35 36 37 38
It is possible that the vasodilator effects of acetylcholine and bradykinin may involve their actions on sympathetic or sensory nerve terminals.23 25 An alternative explanation for the decreased vasodilator potency of acetylcholine in the denervated hindlimb vasculature is that removal of the lumbar sympathetic nerves results in the loss of acetylcholine-induced modulation of sympathetic neurotransmission.23 However, the observation that the vasodilator effects of acetylcholine and bradykinin "use-dependently" decrease in the denervated bed strengthens the likelihood that preformed NOFs exist within the vascular endothelium of this bed. Similarly, bradykinin has been reported to directly stimulate sensory neurons in several species,25 and as such, its effects may be due to the release of vasodilator factors such as an S-nitrosothiol from sensory terminals because NO synthase has been shown to exist in peripheral sensory neurons.4 The use-dependent loss of bradykinin-induced vasodilation may therefore involve the depletion of preformed pools of NOFs within sensory terminals.
The possibility that the use-dependent loss of endothelium-dependent vasodilation in the presence of L-NAME is due to the rapid desensitization of muscarinic and bradykinin receptors deserves to be addressed. To our knowledge, there is no evidence to support this possibility. In vitro findings that removal of the endothelium converts acetylcholine-induced vasodilation to vasoconstriction1 certainly suggest that the function of muscarinic receptors on vascular smooth muscle, at least, is not compromised by the absence of NOFs. Moreover, it seems unlikely that the use-dependent loss of bradykinin-induced vasodilation was due to an L-NAME–induced desensitization of bradykinin receptors because the behavioral effects of this autacoid, which are presumably mediated by activation of bradykinin receptors on sensory afferents,25 were unaffected by the NO synthase inhibitor.
Our observation that chronic surgical sympathectomy resulted in a pronounced vasoconstriction in the ipsilateral hindlimb is consistent with a previous finding that the destruction of sympathetic nerves by neonatal administration of guanethidine resulted in an increase in HLR in adult rats.39 Our observation that acetylcholine produced smaller vasodilator responses in the denervated hindlimb is consistent with the finding that chronic sympathectomy of the rabbit ear artery greatly reduces the endothelium-dependent vasorelaxant effects of the muscarinic agonist methacholine.40 Moreover, our observation that L-NAME produced a relatively minor increase in HLRd is consistent with findings that NO synthase inhibitors produced less vasoconstriction in sympathectomized hindlimbs of the cat.7 Sympathetic nerves play a vital role in the synthesis and release of endothelial NOFs in anesthetized rats.41 Moreover, increases in sympathetic nerve activity resulting from bilateral lesion of the nucleus of the tractus solitarius results in marked catecholamine-induced (and presumably NO-mediated) increases in the cGMP synthesis within vascular tissue.42 The activation of the lumbar sympathetic chain by air-jet stress causes a pronounced hindlimb vasodilation in conscious rats that is reduced by L-NAME.22 Moreover, a subpopulation of postganglionic lumbar sympathetic nerves innervating the rat hindlimb contains NO synthase.22 Taken together, these findings suggest that the hindlimb vasoconstriction produced by chronic lumbar sympathectomy is due to the combination of a loss of endothelial NO synthase activity and the sympathetic neurogenic vasodilator system. It would therefore appear that the loss of these vasodilator systems outweighs the loss of the sympathetic neurogenic vasoconstrictor input to this bed.
In summary, the results of the present study support Ignarro's13 postulation that endothelium-dependent agonists not only initiate the de novo synthesis of NO or NOFs but also the release of preformed pools of NO-containing factors such as S-nitrosothiols11 or dinitrosyl iron (II) complexes.12 We have recently reported that neurogenic vasodilation in the hindlimb of rats may be mediated by the release of preformed neurotransmitter pools of NOFs from NO synthase–positive postganglionic lumbar sympathetic nerve terminals innervating this bed.21 The presence of these preformed pools of NOFs in the vascular endothelium and sympathetic vasodilator nerves would provide an additional mechanism by which these cells can regulate vascular tone, especially under conditions in which NO synthesis is temporarily compromised.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grant HL-14388. The authors wish to thank Mike Burcham for his expert assistance in the preparation of the summary figures shown in this manuscript.
Received February 27, 1996; first decision April 4, 1996; accepted May 13, 1996.
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References |
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